19 research outputs found

    Sensitivity of various CaSR mutants to L-Phe in HEK293 cells.

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    <p>a. HEK-293 cells transfected with CaSR or its mutants were loaded with Fura-2 AM for 15 min. The intracellular Ca<sup>2+</sup> level was assessed by monitoring emission at 510 nm with excitation alternately at 340 or 380 nm using fluorescence microscopy as above. Each experiment started with 1.5 mM mM Ca<sup>2+</sup> followed by stepwise increases in the level of L-Phe (up to 12.0 mM) while the [Ca<sup>2+</sup>]<sub>o</sub> was maintained at 1.5 mM. b. The sensitivity of P221Q and L173P to L-Phe were compared at 5.0 mM [Ca<sup>2+</sup>]<sub>o</sub> or 15.0 mM [Ca<sup>2+</sup>]<sub>o,</sub> respectively.</p

    Summary of EC<sub>50</sub>s of concentration-response curves for L-Phe at different [Ca<sup>2+</sup>]<sub>o</sub>.

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    <p>The [Ca<sup>2+</sup>]<sub>i</sub> responses of HEK-293 cells transfected with CaSR or its mutants upon stepwise increases of L-Phe in the presence of 1.5 mM [Ca<sup>2+</sup>]<sub>o</sub> were recorded. For L173P and P221Q, the L-Phe induced intracellular change were also measured in the presence of a level of high [Ca<sup>2+</sup>]<sub>o</sub> corresponding to their EC<sub>50</sub> values for the [Ca<sup>2+</sup>]<sub>o</sub>, specifically 15.0 mM [Ca<sup>2+</sup>]<sub>o</sub> for mutant L173P and 5.0 mM for P221Q. The results are shown in the brackets. The EC<sub>50</sub>s were calculated from the concentration-response curves fitted using the Hill equation. <sup>#</sup> indicates significance with respect to wild type CaSR, p<0.05 (ANOVA, Dunnett test); <sup>*</sup> indicates significance with respect to the corresponding mutants in the presence of 1.5 mM [Ca<sup>2+</sup>]<sub>o</sub>, p<0.05 (two-way ANOVA).</p><p>Summary of EC<sub>50</sub>s of concentration-response curves for L-Phe at different [Ca<sup>2+</sup>]<sub>o</sub>.</p

    The disease-related mutations are located in the hinge region near Ca<sup>2+</sup>-binding site 1.

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    <p>a. Model structure of CaSR ECD based on the mGluR1 crystal structure (PDB entry: 1ISR) was generated using MODELER 9v4 and Swiss-Model. The global view of the ECD is shown in the left panel. Spheres highlighted in red: Ca<sup>2+</sup>; Purple: residues involved in predicted Ca<sup>2+</sup>-binding sites. L-phenylalanine (in yellow) is positioned at the hinge region between the two lobes by Autodock vina. Right panel: A zoomed in view of the Ca<sup>2+</sup>-binding pocket of site 1. Residues involved in Ca<sup>2+</sup>-binding site 1are highlighted in purple; residues with disease-related mutations are highlighted in pink; residues predicted to interact with L-Phe are presented in green. b. Immunofluorescence analysis of surface expressed WT CaSR and its mutants in HEK293 cells. Immunostaining was done with anti-flag monoclonal antibody, and detection was carried out with Alex Fluor 488-conjugated, goat anti-mouse secondary antibody. Green: CaSR. The images were taken using equal exposure times. c. Schematic figure of calcium and L-Phe induced downstream signaling changes and the principle for the measurement of IP<sub>1</sub> accumulation. Red dots represent calcium ions. d. total proteins (40 µg) were applied to SDS-PAGE under non-reducing conditions and blotted with anti-flag antibody. GAPDH was used as an internal control. e. The signals were analyzed using ImageJ and the normalized intensities were compared with WT CaSR.</p

    Frequency distribution of oscillation parameters in HEK293 cells transfected with CaSR or its mutants.

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    <p>The pattern of the [Ca<sup>2+</sup>]<sub>i</sub> response in each cell (minimum of 40 cells) was analyzed. a. The [Ca<sup>2+</sup>]<sub>o</sub> at which individual cells started to oscillate was recorded. The X-axis comprises 1.0 mM to 8.0 mM [Ca<sup>2+</sup>]<sub>o</sub> for WT and for mutants L173F and P221L, 1.5 mM to 10.0 mM [Ca<sup>2+</sup>]<sub>o</sub> for P221Q or 7.5 mM to 25.0 mM for mutant L173P. b. The frequency of the individual cell oscillation patterns was investigated. For experiments without L-Phe, the peaks per minute were recorded at the levels of [Ca<sup>2+</sup>]<sub>o</sub> at which the majority of the cells (>50%) started oscillating. Specifically, for the gain-of-function mutants, the peaks per minute were recorded at 2.5 mM [Ca<sup>2+</sup>]<sub>o</sub>, while for loss-of-function mutants, the frequency was analyzed at 15.0 mM [Ca<sup>2+</sup>]<sub>o</sub> for L173P and 5.0 mM [Ca<sup>2+</sup>]<sub>o</sub> for P221Q. c. The [Ca<sup>2+</sup>]<sub>o</sub> at which the [Ca<sup>2+</sup>]<sub>i</sub> oscillations began to reach a plateau was recorded. Empty bar: in the absence of L-Phe; Black bar: in the presence of 5 mM L-Phe.</p

    Principal component analysis (PCA) of the CaSR ECDs.

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    <p>The trajectories of the molecular dynamics simulations were analyzed using principal component analysis (PCA), which separates out the motions of the CaSR ECD into principal modes ranked according to their relative contributions. The first three principal modes were included in the present study to analyze the four CaSR mutants and the wild type: WT (black), L173F (green), presence P221L (magenta), L173P (red), P221Q (blue).</p

    Summary of EC<sub>50</sub>s from experiments measuring IP<sub>1</sub> accumulation and ERK<sub>1/2</sub> phosphorylation.

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    <p>HEK293 cells were transiently transfected with the WT CaSR or disease-associated CaSR mutants. Cells were then treated with various levels of [Ca<sup>2+</sup>]<sub>o</sub>. The EC<sub>50</sub>s of [Ca<sup>2+</sup>]<sub>o</sub>-IP<sub>1</sub> responses and the -[Ca<sup>2+</sup>]<sub>o</sub>-ERK<sub>1/2</sub> phosphorylation activity responses were obtained from curve fitting using the Hill equation as mentioned in Method. <sup>#</sup> indicates significance with respect to wild type CaSR, p<0.05 (ANOVA, Dunnett test); <sup>*</sup> indicates significance with respect to the corresponding mutants in the absence of L-Phe, p<0.05 (two-way ANOVA).</p><p>Summary of EC<sub>50</sub>s from experiments measuring IP<sub>1</sub> accumulation and ERK<sub>1/2</sub> phosphorylation.</p

    Schematic representation of the mechanisms underlying the effects of the mutations on the CaSR and the modulation of receptor activity by extracellular Ca<sup>2+</sup> and L-Phe.

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    <p>Ca<sup>2+</sup> and L-Phe modulate the activity as well as the cooperativity of CaSR (the color changes of the receptor from white to red indicates an increase in functional activity). Elevating [Ca<sup>2+</sup>]<sub>o</sub>, e.g., to 3.0 mM, is proposed to change the basal CaSR status into an active form in a positive homotropic cooperative manner and further trigger [Ca<sup>2+</sup>]<sub>i</sub> oscillations. L-Phe binds to the hinge region between lobe 1 and lobe 2, modulating the receptor together with Ca<sup>2+</sup> in a positive heterotropic cooperative way. This could potentiate conversion of the receptor to a “fully active” form associated with a higher frequency of [Ca<sup>2+</sup>]<sub>i</sub> oscillations and a left-shifted EC<sub>50</sub>. Loss-of-function CaSR mutants (indicated by white color) could cause a disruption of the cooperativity among the various Ca<sup>2+</sup>-binding sites (dashed arrows). [Ca<sup>2+</sup>]<sub>o</sub> at 3.0 mM does not trigger [Ca<sup>2+</sup>]<sub>i</sub> oscillations in the mutant CaSRs. The impaired receptor function and the cross-talk between Ca<sup>2+</sup>-binding sites can be at least partially rescued for some mutants by L-Phe (e.g.P221Q). However, if the mutation interferes the interaction between CaSR and L-Phe, the function of the receptor may not be fully recovered (e.g.L173P). CaSR gain-of-function mutants (left) exhibit enhanced correlated motions (double line arrows) and their activity is not further potentiated by L-Phe, potentially due to a ceiling effect.</p

    Functional studies of disease-related CaSR mutations in individual HEK293 cells.

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    <p>The various panels show representative oscillation patterns from single cells. HEK-293 cells transfected with CaSR or one of its mutants were loaded with Fura-2 AM for 15 min. [Ca<sup>2+</sup>]<sub>i</sub> was assessed by monitoring emission at 510 nm with excitation alternately at 340 or 380 nm as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113622#s2" target="_blank">Methods</a>. Each experiment was carried out with or without 5 mM L-Phe and began in Ca<sup>2+</sup>-free Ringer's buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, and 1.0 mM MgCl<sub>2</sub>, pH 7.4), followed by stepwise increases in [Ca<sup>2+</sup>]<sub>o</sub> until [Ca<sup>2+</sup>]<sub>i</sub> reached a plateau (up to 30 mM [Ca<sup>2+</sup>]<sub>o</sub>).</p

    L-Phe potentiates [Ca<sup>2+</sup>]<sub>o</sub>-activated ERK signaling in CaSR-transfected HEK293 cells.

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    <p>HEK-293 cells transfected with WT CaSR or its mutants were incubated in serum-free high glucose MEM medium containing 0.2% BSA overnight. Cells were washed with Hank's balance salt solution (HBSS) and then incubated in the presence of various Ca<sup>2+</sup> concentrations (0.0-∼25.0mM) in the absence or presence of 5 mM L-phenylalanine for 10 min at 37°C. The incubations were stopped by exposure to the lysis buffer and processed for SDS/PAGE and Western blotting as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113622#s2" target="_blank">Methods</a>. The Western blot results were further quantified using Image J. All [Ca<sup>2+</sup>]<sub>o</sub>-concentration response curves were normalized to the maximum response in each individual experiment. The Hill equation was employed to fit the data. Markers: [Ca<sup>2+</sup>]<sub>o</sub> only; closed markers: with L-Phe.</p

    Summary of cellular responses of HEK293 cells transiently transfected with WT CaSR or disease related mutants.

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    <p>The average levels of [Ca<sup>2+</sup>]<sub>o</sub> at which cells started to exhibit [Ca<sup>2+</sup>]<sub>i</sub> oscillations were recorded for WT or each mutant CaSR. For the oscillation frequencies in the absence of L-Phe, peaks per minute were measured at the level of [Ca<sup>2+</sup>]<sub>o</sub> at which more than 50% cells started to oscillate; when L-Phe was added, frequencies were recorded at the same [Ca<sup>2+</sup>]<sub>o</sub> as their counterparts without L-Phe. Specifically, the frequencies of WT was measured at 3.0 mM [Ca<sup>2+</sup>]<sub>o</sub>, at 2.5 mM [Ca<sup>2+</sup>]<sub>o</sub> for L173F and P221L; at 15.0 mM [Ca<sup>2+</sup>]<sub>o</sub> for L173Pand at 5.0 mM [Ca<sup>2+</sup>]<sub>o</sub> for P221Q. Curve-fitting was performed using the Hill equation. The data were obtained from three experiments for each construct. Values are means ± S.E. EC<sub>50</sub> and Hill numbers obtained from the cell population assay by fitting plots using the Hill equation. <sup>#</sup> indicates significance with respect to wild type CaSR in the absence of L-Phe, p<0.05 (ANOVA, Dunnett test); <sup>*</sup> indicates significance with respect to the corresponding mutants in the absence of L-Phe, p<0.05 (two-way ANOVA).</p><p>Summary of cellular responses of HEK293 cells transiently transfected with WT CaSR or disease related mutants.</p
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